ABSTRACT Bone adapts its structure to mechanical loading. This adaption is essential for growing the right skeleton and maintaining its integrity throughout life. Osteocytes are the cells responsible for sensing and coordinating response to mechanical load. Key recent discoveries reported by our group during the last several years established that osteocyte cell processes function as unique mechanosensory elements. Processes are >10-fold more sensitive to mechanical stimuli than osteocyte cell bodies. Moreover, this triggering of Ca2+ signaling from cell processes occurs through a unique complex of aVb3 integrins, membrane channels and receptors, that occur at attachment points to the canalicular walls, and which we call the ?Osteocyte mechanosome.? This proposal is based on the global hypothesis that a novel structure localized on osteocyte processes, the osteocyte mechanosome, detects and transduces mechanical signals. To date, we have identified four key osteocyte mechanosome components: ?V?3 integrin, pannexin1, P2X7 receptor (P2X7R) and the CaV3.2 T-type calcium channel. Our multidisciplinary team will test this hypothesis by multiple approaches in each of three aims. In Aim 1 we will combine biochemical techniques (co-immunoprecipitation, surface plasmon resonance) and imaging modalities (FRAP, FRET and STORM super-resolution microscopy) to define comprehensively the structural and dynamic properties of this heretofore unknown transduction complex, the osteocyte mechanosome in osteocytic cells in vitro. In Aim 2 we test how pharmacological and genetic alteration of individual mechanosome components alters upstream (Ca2+) and downstream (to bone) signaling in osteocytic cells in vitro. In Aim 3, we will combine our novel OtGP3 osteocyte Ca2+ reporter mice-in vivo loading/imaging system with pharmacological manipulations to confirm effects of key mechanosome components (as identified in Aims 1 and 2) on osteocyte Ca2+ response and on downstream signaling. We will also use this approach to answer the fundamental question of whether osteocyte Ca2+ responses to mechanical loading altered by loss of constitutive sex hormones (estrogen/androgen) or by anabolic PTH.